2.1 LECTIN
Lectins are oligomeric sugar-binding proteins found in plant and animal cells as well as microorganisms and viruses. Although most lectins are glycoproteins, several lectins such as Concanavalin A, peanut lectin, and wheat germ agglutinin lack covalently bound carbohydrates and it is believed that the bound sugars do not play a significant role in the binding activity of lectin glygcoproteins.
At present, the biological role of lectin protein in nature is unclear. However, these proteins have been used extensively in biological research and medical diagnostics. The utility of lectins derives from their ability to bind reversibly to specific carbohydrate moieties without altering them chemically. Since each lectin binds specifically to a particular carbohydrate structure (e.g., Phaseolus vulgaris lectin binds to N-acetylgalactosamine linked to mannose), lectins provide a highly specific method for distinguishing and isolating oligosaccharides, glycoproteins and cells carrying these carbohydrate structures on their surfaces. Thus, lectins have been used to precipitate polysaccharides and glycoproteins, to agglutinate cells such as erythrocytes of specific blood groups (i.e., in blood typing) and to separate cancerous cells from normal cells. In addition, lectin affinity chromatography (in which lectin is covalently bound to a column matrix such as agarose) allows the separation and purification of soluble glycoproteins, glycopeptides, polysaccharides and glycosylated nucleic acids.
Another property of some lectins is their ability to act as mitogens, inducing mitosis in cultured cells. This effect is due in part to the protein's binding to the cell surface, although other cell surface interactions are believed to be involved as well.
Lastly, lectins are more toxic to cancerous cells in culture than to normal culture cell lines and have been used in the design and construction of new selective toxins.
Although plant lectins have been the most widely studied [see Lis and Sharon, Annu. Rev. Biochem. 42: 541 (1973)], lectins have been isolated from various animal sources including snails [see Cohen, et al., Life Sci. 4: 2009 (1965)], fish [see Pardoe and Uhlenbruck, J. Med. Lab. Technol. 27: 249 (1970)]and rabbits [see Lurrey and Ashwell, Proc. Natl. Acad. Sci., U.S.A. 73: 341 (1976)]. For example, mammalian hepatic lectin found in rabbit liver is belived to remove certain plasma proteins from the blood and has been shown to act as a mitogen for lymphocytes. In addition, lectin protein has also been found in fungi [see Sage and Corrett, J. Biol. Chem. 244: 4713 (1969)].
Although lectins vary in their carbohydrate specificity, cell binding and agglutinating ability and subunit arrangement, there appears to be a significant degree of homology between the amino acid sequences, overall molecular weights, subunit molecular weights and subunit numbers of various lectins from leguminous plants [see Foriers, et al., Physiol. Veg. 17(3): 597 (1979)]. In addition, animal lectins possess a subunit molecular weight and amino acid composition similar to that of plant lectins [see Brown and Hunt, Int. Rev. Cytol. 52: 277 (1978); Sharon and Lis, Science 177(4053): 949 (1972)].
The conventional methods for purification of lectins involved standard procedures of protein fractionation including ethanol precipitation, salt-induced crystallization, ion exchange chromatography and gel filtration. However, lectin purification is particularly difficult using standard procedures. Many lectins are composed of several subunits and these often become dissociated during extraction. In addition, the subunits may reassociate to form various permutations of the protein. Another problem is the physiochemical similarity between the various lectins making purification of a specific lectin difficult.
Increasingly, affinity chromatography has been utilized to isolate and purify lectins. A protein extract is passed over a column packed with an inert matrix to which a specific carbohydrate structure has been attached. The lectin specific for that carbohydrate structure binds to the moiety and is retained on the column while the rest of the protein extract is eluted off the column with buffer. To remove the lectin from the column, sugar solutions that inhibit lectin binding are utilized as eluents.